The therapeutic potential of bispecific antibodies has long been recognized. Bispecific antibodies offer an IgG like platform that is able to bind two antigens or two epitopes simultaneously. Thus, bispecific antibodies offer a potential tool to modulate the interaction of at least two molecules and/or the interaction of at least two systems comprising the molecules. Such modulation may be, for example, modulation of the interaction of two cells where the recognized antigen, antigens and/or epitopes are expressed on the surface of the cells. Examples of the therapeutic use of bispecific antibodies include, for example, the modulation of cell signaling (e.g., by promoting or interfering with the interaction of desired surface receptors or ligands) and cancer therapies (e.g., aiding in the targeting of immune cells to cancer cells).
Despite the interest in the therapeutic use of bispecific antibodies, their commercial production has proven problematic. Early attempts focused on the fusion of two hybridoma cell lines each expressing monospecific, bivalent antibodies (“quadroma technology”, see, e.g., Milstein and Cuello, Nature 305(1983), 537-540). Although the quadroma expressed antibody molecules, it was immediately apparent that the expressed molecules contained varying combinations of the two parental heavy and two parental light chains. The simultaneous expression of all four parental chains lead to a mixture of 10 different variants of almost identical molecules, wherein only 1 of the 10 (i.e., only a minor fraction of all molecules expressed) contained the properly paired heavy and light chains necessary to exhibit the desired bispecific activity (see, e.g., Suresh et al., Methods Enzymol. 121(1986), 210-228). Accordingly, attention turned to alternate bispecific antibody-based constructs in an attempt to eliminate the production problems, e.g., single-chain fusions of antibody variable domains. However, many of these formats differ significantly from the archetypical antibody structure and were found to exhibit therapeutic disadvantages such as poor pharmacokinetic properties and/or loss of effector activity (e.g., due to absence of Fc domains). Further, many constructs also exhibited a tendency to aggregate and an increased potential for immunogenicity due to the presence of non-human or artificial domains such as linker regions.
In view of the limitation of alternate bispecific formats, and in spite of the production difficulties, interest in bispecific antibodies having the archetypical antibody architecture remains (in particular, IgG like architecture). Principally, two problems arise during the production of a desired bispecific antibody having IgG like architecture. Because such a molecule requires the proper association of 2 different heavy chains and 2 different light chains, it is necessary (1) to induce hetero-dimerization of the two different heavy chains as a preferred reaction over homo-dimerization, and (2) to optimize the discrimination among the possible light-chain/heavy-chain combinations interactions such that the expressed molecule contains only the desired light-chain/heavy-chain interactions. These two issues have effectively been solved.
First, the hetero-dimerization of the two different heavy chains has been shown to be promoted over homo-dimerization interactions by the use of “knobs into holes” or “KiH” methodology. In KiH methodology, large amino acid side chains are introduced into the CH3 domain of one of the heavy chains, which side chains fit into appropriately designed cavities in the CH3 domain of the other heavy chain (see, e.g., Ridgeway et al., Protein Eng. 9(1996), 617-621 and Atwell et al., J. Mol. Biol. 270(1997), 677-681). Thus, heterodimers of the heavy chains tend to be more stable than either homodimer, and form a greater proportion of the expressed polypeptides.
Second, the association of the desired light-chain/heavy-chain pairings can be induced by modification of one Fab of the bispecific antibody (Fab region) to “swap” the constant or constant and variable regions between the light and heavy chains. Thus, in the modified Fab domain, the heavy chain would comprise, for example, CL-VH or CL-VL domains and the light chain would comprise CH1-VL or CH1-VH domains, respectively. This prevents interaction of the heavy/light chain Fab portions of the modified chains (i.e., modified light or heavy chain) with and the heavy/light chain Fab portions of the standard/non-modified arm. By way of explanation, the heavy chain in the Fab domain of the modified arm, comprising a CL domain, does not preferentially interact with the light chain of the non-modified arm/Fab domain, which also comprises a CL domain (preventing “improper” or undesired pairings of heavy/light chains). This technique for preventing association of “improper” light/heavy chains is termed “CrossMab” technology and, when combined with KiT technology, results in remarkably enhanced expression of the desired bispecific molecules (see, e.g., Schaefer et al., PNAS 108(2011), 11187-11192). Alternately or additionally, one arm of the antibody may be modified such that the Fab domain is a scFab or scFv, leaving only one “free” light chain in the system.
Despite the recent advantages in the expression of bispecific antibodies, use of the molecules remains constrained due to the formation of byproducts specifically associated with their production (bispecific-antibody specific byproducts, “BASB”) and the problems associated with BASB separation from the desired molecules. As compared to the purification of standard antibodies, the economic purification of bispecific antibodies from production media represents unique challenges. The production of a standard antibody relies on the dimerization of identical heavy-chain/light-chain subunits. In contrast, the production of a bispecific antibody requires the dimerization of two different heavy-chain/light-chain subunits, each comprising a different heavy chain as well as a different light chain. Thus, bispecific antibody production requires the proper interaction of up to four peptide chains. Accordingly, chain mispairings (e.g., homo-dimerization of identical heavy chain peptides or improper heavy-chain/light-chain associations) are often observed, as is incomplete protein assembly due to unbalanced expression of the different antibody chains. Commonly observed BASB include ½ antibodies (comprising a single heavy-chain/light-chain pair) and ¾ antibodies (comprising a complete antibody lacking a single light chain). Additional BASB may be observed depending on the bispecific format used. For example, where one variable domain of the bispecific antibody is constructed as a single-chain Fab (scFab), a 5/4 antibody by-product (comprising an additional heavy or light chain variable domain) may be observed. Such corresponding byproducts are not normally seen in standard antibody production.
Moreover, BASB may exhibit particularly disadvantageous activity should they remain in the final purified product. With respect to standard antibodies, i.e., monospecific antibodies, it can be seen that the above-described by-products contain at least one functional antigen binding site. Therefore, such a byproduct in a monospecific antibody formulation would likely be partially if not entirely therapeutically functional and, thus, of little concern in any purification scheme. In contrast, BASB represent impurities that, depending on bispecific format, could negatively impact the activity of the desired bispecific formulation. Thus, their separation from the desired molecule during purification becomes critical. For example, the functionality of the bispecific molecule may depend on a single molecule exhibiting binding activity to two different antigens. Where a molecule exhibits binding activity to only one target antigen (e.g., as in a ½ or ¾ antibody as described above), its binding to this target antigen would block the binding of a fully functional bispecific antibody, potentially antagonizing the desired activity of the bispecific molecule. At the very least, the monospecific byproducts of bispecific antibody production would likely reduce efficacy of the final bispecific formulation if not separated. Additionally, many of the BASB as described herein, having exposed regions that normally promote peptide-peptide interaction, exhibit a tendency to immunogenicity and aggregation.
Unfortunately, most commercial antibody production and purification schemes are unsuitable or incapable of separating bispecific antibodies from the above-described specific byproducts. Standard antibody purification schemes usually involve at least two distinct modes of chromatography, that is, usually employ at least two chromatographic mechanisms to separate the desired immunoglobulin(s) from byproducts/impurities. The first mode is usually an affinity-based chromatography that utilizes a specific interaction between the protein to be purified (i.e., the protein of interest) and an immobilized capture reagent. Because affinity reagents may represent the most expensive portion of a purification scheme, it is desirable to reduce the use of affinity ligands and/or to maximize applicability of a particular scheme (and affinity reagent) across a number of products. The most commonly used affinity ligand in immunoglobulin purification (and applicable to a wide range of immunoglobulin-based products) are the Fc-binding or constant domain-binding agents such as Protein A, Protein G, Protein L, KappaSelect™ and LambdaFabSelect™. However, the separation activity of these Fc- or constant domain-binding agents is based on the presence of an Fc-, κ-, and/or λ-domains, which, importantly, are shared by the bispecific antibody and its specific byproducts (i.e., BASB). Accordingly, Fc- or constant domain-affinity ligands alone are insufficient to purify bispecific antibodies from the BASB, and the implementation of additional affinity based purifications and/or molecule specific (i.e., antigen-specific) purification would likely lead to economically prohibitive schemes.
Further, based on the understanding in the art prior to the present invention, the addition of other common purification processes used in commercial antibody processing schemes would also not be believed sufficient to satisfactorily separate bispecific antibodies from BASB. The most common purification processes used in conjunction with affinity chromatography for commercial antibody purification are standard chromatography methods that separate the protein of interest from undesired byproducts/impurities based on differences in size, charge (e.g., isoelectric point or “IEP”), solubility, and/or degree of hydrophobicity. Such methods include ion exchange chromatography, size exclusion chromatography, immobilized metal affinity chromatography, and hydroxyapatite chromatography. However, these common chromatography processes using standard protocols are unsuitable for the separation of bispecific antibody from BASB: size exclusion chromatography is not economically feasible for large-scale purifications and the differences in IEP between the bispecific antibody and BASB were believed in large part too small for their separation by ion-exchange chromatography.
Therefore, known methods for the separation of antibodies from solutions comprising by-products specific to their production (e.g., Fc containing antibody fragments) are ineffective for the purification of bispecific antibodies and/or may be undesirable for economic reasons (e.g., the use of additional affinity chromatography steps). Accordingly, there is a need for new and/or improved schemes for the purification of bispecific antibodies from production solutions (and, in particular from BASB contained therein), which schemes are able to meet the requirements of the biotechnology industry for the production of diagnostic and therapeutic products (e.g., demonstrating effective cost, throughput and product purity).